1. Field of the Invention
The present invention relates to methods of producing polypeptides having biological activity in bacterial host cells. The present invention also relates to isolated modified mRNA processing/stabilizing sequences and to nucleic acid constructs, vectors, and host cells comprising the modified mRNA processing/stabilizing sequences operably linked to promoter regions for expressing polynucleotide sequences encoding polypeptides having biological activity.
2. Description of the Related Art
The recombinant production of a native or heterologous polypeptide having biological activity in a bacterial host cell, particularly a Bacillus cell, may provide for a more desirable vehicle for producing the substance in commercially relevant quantities.
Recombinant production of a native or heterologous polypeptide having biological activity is accomplished by constructing an expression cassette in which the DNA coding for the polypeptide is placed under the expression control of a promoter, excised from a regulated gene, suitable for the host cell. The expression cassette is introduced into the host cell, usually by plasmid-mediated transformation. Production of the polypeptide is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
Translation initiation frequency, codon usage, and RNA secondary structure are factors that affect mRNA stability in bacteria (Regnier and Arraiano, 2000, Bioessays 22: 235-244; Steege, 2000, RNA 6: 1079-1090). In Bacillus subtilis, mRNA stability is influenced by ribosome stalling at the 5′-untranslated region. For example, the stability of the Bacillus subtilis alkaline protease gene (aprE) was found to have a high half-life in Bacillus subtilis of approximately 25 minutes as a result of the ribosome binding site (Shine-Dalgarno sequence) present in the gene leader region (Hambraeus et al., 2002, Microbiology 148: 1795-1803). The complementarity of the Shine-Dalgarno sequences at the 3′-OH end of the 16S ribosomal RNA determines the affinity between the ribosomal subunits and the mRNA. The fixation of a ribosomal subunit to the 5′-UTR has been reported to be an mRNA stability inducer in Bacillus subtilis (Sharp and Bechhofer, 2003, J. Bacteriology 173: 4952-4958). The presence of Shine-Dalgarno-like sequences, referred to as stabilizer sequences, in the 5′-UTR has been proposed to be one of the causes of high mRNA stability observed for the cryIIIA gene (Agaisse and Lereclus, 1996, Mol. Microbiol. 20: 633-643). U.S. Pat. Nos. 6,255,076 and 5,955,310 describe the use of the cryIIIA mRNA stabilizer sequence for improved expression of enzymes in Bacillus cells. Daguer et al., 2005, Letters in Applied Microbiology 41: 221-226, describe increasing the stability of sacB transcripts improves levansucrase production in Bacillus subtilis. 
It would be an advantage in the art to provide new methods for transcript stabilization to improve the level of a polypeptide expressed by a bacterial host strain.
The present invention relates to improved methods of producing a polypeptide having biological activity in a bacterial host cell.